The sentinel physicians are asked to take nose and/or throat swabs from patients with ILI or ARI. Some sentinel surveillance systems also collect blood samples (e.g. the Czech Republic, Romania and the Slovak Republic). The specimens are sent to the national reference laboratory and are tested for influenza viruses (if positive, subtypes are determined) and other respiratory viruses. These results are used to validate the clinical reports of ILI and ARI. View table

The National Reference Laboratories also report influenza test results from non-sentinel surveillance physicians to EISN. Specimens (nose swabs, throat swabs and blood samples) can come from a wide range of sources: hospitals, non-sentinel physicians, homes for the elderly, clinics, etc. These data are collected to validate the data provided by the sentinel surveillance systems and to better describe the epidemiology and virology of influenza in each network.

Tests performed by the Community Network of Reference Laboratories (CNRL)

To guarantee the reporting of accurate virological data to EISN, laboratories participating in the CNRL must be able to perform a range of basic tasks. 

Abbreviations

CFT	complement fixation test
ELISA	enzyme-linked immunosorbent assay
IF	immunofluorescence
HI	hemagglutination inhibition assay
NAT	nucleic acid amplification techniques
WHO-CC	WHO-Collaboration Centre for Reference and Research on Influenza

Direct detection of influenza virus A and B

Detection, typing and/or subtyping of influenza virus in a clinical specimen can be done simultaneously using type and/or subtype specific tests. These tests can be genetic (by NAT) or antigenic (IF or ELISA).

Culture of influenza virus

Laboratories must be able to isolate and grow influenza on cell lines. Growth of influenza virus in embryonated eggs (required for the bi-annual selection of vaccine candidate viruses) is not strictly necessary for virus detection and is considered a secondary requirement. Cultured virus is detected using the techniques described above and by determining the hemagglutinating activity of the culture supernatant or egg allantoic fluid. The use of the WHO identification kit using sheep antisera for the HI assay is recommended for the identification of isolates. At the start of each season, this kit is distributed to all CNRL laboratories by the WHO-CC located at the CDC in Atlanta, US. The WHO-CC also distributes identification kits for emerging viruses, such as the A(H5N1) or A(H1N1)v virus, to the CNRL laboratories on request.

Determination of influenza virus type and subtype

CNRL members are required to type influenza virus A and B and to subtype both the hemagglutinin (H1, H2, H3, H5, H7, H9 and H10) and neuraminidase (N1, N2, N3 and N7) of type A influenza viruses that are currently circulating, have previously caused a pandemic or are of avian origin causing human infection. Typing and subtyping can be done antigenically (e.g. by HI) or genetically (by type- and subtype-specific NAT).

Antigenic and genetic characterisation of influenza virus

Antigenic characterisation of the hemagglutinin is required to check the match of the seasonal vaccine with circulating viruses and for the selection of vaccine candidate viruses. Antigenic characterisations are carried out using ferret antisera and reference virus strains distributed by the WHO-CC in London, with which an agreement was signed for the delivery of these reagents to the CNRL. A representative set of isolates is sent during the season to the WHO-CC in London that will perform characterisations for CNRL laboratories unable to do so, confirm characterisations and compare isolates with viruses from all over the world to facilitate WHO decision-making on the seasonal vaccine composition. Genetic characterisation is performed by sequencing and this sequence information is shared with the WHO-CC in London. Although information on genetic drift can not directly be translated into vaccine match, sequence information contributes to WHO decision-making on the seasonal vaccine composition.

Diagnostic influenza serology

This is performed by using HI or CFT with paired sera where possible. These techniques are particularly important for retrospective analysis of outbreaks, which might take place nationally, at a closed institution, a school, a hospital or a geriatric facility. In addition, these techniques are important for retrospective analysis of outbreaks as the result of animal (particularly avian) influenza viruses transmitting to humans during animal influenza epizootics.

Storage of clinical samples and virus isolates

Samples and isolates are stored at or below -70°C for future analysis or usage. If stored samples are later to be tested for the presence of RSV, sucrose should be added before freezing. For additional tips on storage of influenza samples, see the WHO Manual on Animal Influenza Diagnosis.